Purpose To explore the specific diagnostic markers of Sertoli cells and germ cells for testicular biopsy. Methods Normal testis from 3 patients who suffered from carcinoma of the prostate and treated with testis castration, and 15 testicular biopsy tissues were stained by EnVision two steps with WT-1, AR and Ki-67. Results The expression of WT-1 and AR protein were found in Sertoli cells from the 3 normal testis and 15 testis biopsy, the positive rate were all 100%, and non was positive in germ cells. The germ cell was positive for Ki-67, and the positive rate was 100%. Conclusions WT-1, AR and Ki-67 may be the specific diagnostic markers of Sertoli cells and germ cells for testicular biopsy, which may contribute to the diagnosis of testicular biopsy.